The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium traditionally characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. toxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. toxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4-S7; Beegle, C. C., T. Yamamoto, "History of Bacillus thuringiensis Berliner research and development," Can. Ent. 124:587-616). Thus, isolated B.t. toxin genes have increasing commercial value.
Until fairly recently, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline .delta.-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
Investigators have now discovered B.t. pesticides with specificities for a broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; and Beegle, C. C. (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508 describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni.
More recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were cryI (Lepidoptera-specific), cryII (Lepidoptera- and Diptera-specific), cryIII (Coleoptera-specific), and cryIV (Diptera-specific).
The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne L. Kim [1992] Bio/Technology 10:271-275). cryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbiol 62(1):80-86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. U.S. Pat. No. 5,273,746 discloses several B.t. isolates, including PS192M4, as having activity against lice. Gleave et al. ([1991] JGM 138:55-62), Shevelev et al. ([1993] FEBS Lett. 336:79-82; and Smulevitch et al. ([1991] FEBS Lett. 293:25-26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897.). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose B.t. toxins having activity against lepidopterans.
U.S. Pat. Nos. 5,262,159 and 5,468,636 disclose B.t. isolates PS157C1, PS86A1, and PS75J1 for use against aphids. U.S. Pat. Nos. 5,277,905 and 5,457,179 disclose the use of B.t. isolate PS50C for use against coleopteran pests. U.S. Pat. No. 5,366,892 discloses the sequence of the 50C(a) B.t. toxin. U.S. Pat. No. 5,286,485 discloses the use of PS50C against lepidopteran pests. U.S. Pat. No. 5,185,148 discloses the use of PS50C against scarab pests. U.S. Pat. No. 5,554,534 discloses the sequence of the 50C(b) B.t. toxin. U.S. Pat. Nos. 5,262,158 and 5,424,410 disclose the use of PS50C against acarides.
As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
Insects belonging to the order Homoptera include piercing and sucking insects such as leafhoppers and planthoppers. Leafhoppers and planthoppers share a close evolutionary relationship. Leafhoppers and planthoppers are found worldwide and cause serious economic loss to crops and ornamental plants via feeding damage and disease vectoring. A specific example of a planthopper is the brown rice planthopper (Nilaparvata lugens). Because of their piercing and sucking feeding habits, planthoppers and leafhoppers are not readily susceptible to foliar applications of Bacillus thuringiensis (B.t.) proteins in their native, crystal states.